This proposal is designed to provide a comprehensive view of the development of motility by mammalian sperm during epididymal transit. It is based upon existing knowledge of the roles of calcium ion, cyclic AMP, and pH in regulating motility in mature and immature sperm and on our demonstration that adenosine may play a key role in stimulating motility in both mature and immature sperm by virtue of its ability to lower cytosolic levels of calcium ion. We provide evidence in the proposal which allows the description of a sequence of biochemical events in bovine caudal sperm leading from adenosine to the elevation of cyclic AMP levels and the onset of motility. We will test the hypothesis that adenosine stimulates motility in mature bovine sperm by lowering the level of cytoplasmic calcium ion and that this in turn stimulates adenylate cyclase, inhibits cyclic AMP phosphodiesterase, elevates cyclic AMP levels, increases protein phosphorylation, and alters the phosphorylation of CaM-dependent protein kinase substrates. We will also test the hypothesis that bovine caput sperm are immotile because this sequence of events cannot occur. This view is consistent with data we have obtained which shows that bovine caput sperm contain exceptionally high levels of calcium ion (0.8-0.9muM), low levels of adenosine and cyclic AMP and a pHi some 0.4 pH units lower than motile caudal sperm. We will test this view by manipulating, one at a time, the intracellular levels of adenosine, calcium, cyclic AMP, and pHi in caput sperm while simultaneously measuring the levels of all other factors and monitoring sperm motility. In this and all studies in the proposal we will monitor both sperm head and flagellar movement parameters by a fully computerized method developed in our laboratory during the last funding period. In the final stages of the project we will test the hypothesis that the function of the bovine epididymis in the development of sperm motility is to progressively alter, by interaction of sperm and androgen-dependent secretory proteins of epididymal principal cells, the levels of the above factors toward the levels found in mature sperm. To do this we will first determine the levels of all factors in successive segments of the epididymis and correlate these levels with complete motility profiles. Subsequently, we will coculture epididymal principal cells, previously cultured in the presence and absence of androgens, from epididymal regions where significant changes in the levels of calcium, adenosine, cyclic AMP, pHi or the pattern of motility occur and determine whether isolated sperm under the influence of epithelial cell secretory proteins undergo the same changes in culture as were noted in the intact epididymis. If this is found to be so, we will isolate, purify, and characterize these proteins by taking advantage of known, predetermined functions. Successful execution of the aims described in this proposal will lead from a new description of the mechanisms of motility in mature sperm to a new view of how sperm become motile in the epididymis.